Decaprenylamine derivatives

ABSTRACT

This invention relates to new decaprenylamines and the acid addition salts thereof, which are useful for controlling virus infection of vertebrate animals.

There are known heretofore various substances, which have been decidedto have preventive or alleviative effects on diseases caused by viruswhose host is a vertebrate animal, or which have been recognized to becapable of alleviating symptoms of the diseases by significantlyenhancing antibody activity in the animal. Antivirotics reported so farinclude interferon, substances capable of inducing interferon, i.e.inducers (interferon inducers), amantadine hydrochloride or syntheticsubstances, such as methysazone, which directly exert inhibitory effecton the virus propagation. Interferon is glycoprotein having antiviraland antitumor activity, said glycoprotein being produced in situ bycells of a vertebrate animal when the cells are infected with virus, andhas been suggested for the therapy of infectious viral disease and alsofor the therapy of cancer. Known inducers, which induce interferon invertebrate animals by a process other than the virus infection, includenatural high molecular substances such as double chain ribonucleic acidof bacteriophage of a certain species, or synthetic high molecularsubstances such as double chain ribonucleic acid, typical of which ispolyinosinic acid-polycytidylic acid, or low molecular inducers such astyrolone.

In the production of interferon, however, there is involved a problemhow to carry out the purification thereof, and in fact no economicalprocess for the production thereof has not been established yet. On theother hand, conventional interferon inducers have not been put topractical use mainly because of toxicity thereof. Synthetic antiviralagents which directly exert inhibitory effect on the virus propagation,which are commercially available at present, have a rather narrow rangeof virus-infected diseases which are curable by administration of saidagent, and thus the advent of novel synthetic antiviral agents isearnestly desired. Taking such circumstances into consideration, thepresent inventors extensively conducted studies in finding compoundscapable of producing interferon of high potency and, moreover, havingantiviral activity on the biological level, and as the result they haveeventually found that compounds represented by the following generalformula (I) and acid addition salts thereof show excellent interferoninducing ability and, at the same time, demonstrate excellent antiviralactivity even in the biological test.

Thus, the present invention is to provide a new class of adecaprenylamine derivative represented by the following general formula##STR1## wherein n represents an integer of 0-2, R₁ represents hydrogenatom, a lower alkyl group or decaprenyl group, and R₂ represents aphenyl group or a pyridyl group, and acid addition salts thereof.

For the production of decaprenylamine represented by the above-mentionedgeneral formula (I) and acid addition salts thereof, there may beadopted a process in which the known procedures for the amine synthesisare applied to the starting decaprenol represented by the formula##STR2## to produce a desired amine derivative.

Further, the amine derivative thus obtained may be converted into acorresponding salt in the usual way. More specifically, a desired aminecan be produced according to a process which comprises converting asuitable decaprenyl alcohol of the aforesaid general formula (II) into acorresponding halide or sulfonic acid ester, followed by reaction withan appropriate primary or secondary amino compound corresponding to thedesired final product in the presence or absence of a base.Alternatively, the desired amine can be produced by the oxidation of adecaprenol to a corresponding aldehyde, which is then condensed with anappropriate primary amino compound, with splitting off of water, to forma corresponding imino compound which in turn is reduced with a suitablereducing agent (e.g. sodium borohydride). An acid addition salt of theamine derivative thus obtained can be obtained by mixing said amine inan appropriate solvent with a desired acid to form a salt andcrystallizing the salt out of the solution by evaporation or other meansto recover the same. The acid addition salts suitable for use asmedicines include, for example, those with hydrochloric acid, aceticacid, citric acid, fumaric acid and the like.

The compounds represented by the general formula (I) and acid additionsalts thereof are illustrated below with reference to preparativeexample.

PREPARATIVE EXAMPLE 1 N-benzyl-didecaprenylamine hydrochloride ##STR3##

To a solution of benzylamine (10 g.) in ethanol (100 ml) a solution ofdecaprenyl bromide (20 g.) in benzene (40 ml) was added dropwise at roomtemperature for 1 hour with stirring, which was continued for further 2hours. Thereafter, the resulted mixture was heated at reflux for 2 hourswith stirring. The resulting reaction mixture after cooling was addedwith a 2 N sodium hydroxide aqueous solution (100 ml) and then extractedwith isopropylether. The liquid extract obtained was washed with waterand saturated saline, dried over anhydrous sodium sulfate and thenconcentrated under reduced pressure. The residue (21.4 g.) was purifiedby column chromatography using silica gel (200 g.). Elution was carriedout with a mixture of isopropylether and benzene. The initially elutedfraction (7.0 g.) was dissolved in ethyl acetate, added with ethercontaining HCl to weakly acidic and then cooled. The crystallized masswas separated by filtration to recover N-benzyl-didecaprenylaminehydrochloride (4.9 g.), m.p. 52°-54° C. Elementary analysis as C₁₀₇H.sub. 169 N.HCl showed the following:

    ______________________________________                                                C %         H %    N %                                                ______________________________________                                        Calcd.:   85.34         11.38  0.93                                           Found:    85.08         11.23  0.94                                           ______________________________________                                    

PREPARATIVE EXAMPLE 2 N-benzyl-decaprenylamine hydrochloride ##STR4##

The lastly eluted fraction (7.3 g.) obtained in Preparative Example 1was dissolved in acetone and then added with ether containing HCl. Themixture was worked up in the same manner as in Example 1, thereby toobtain N-benzyl-decaprenylamine hydrochloride (5.8 g.), m.p. 105°-107°C. Elementary analysis as C₅₇ H₈₉ N.HCl showed the following:

    ______________________________________                                                C %         H %    N %                                                ______________________________________                                        Calcd.:   83.00         11.00  1.70                                           Found:    82.82         10.87  1.65                                           ______________________________________                                    

PREPARATIVE EXAMPLES 3 TO 9

The same procedures as in Example 1 were carried out for the reaction ofdecaprenyl bromide with a primary or secondary amino compound thereby toproduce the below-indicated compounds, the structural formula, molecularformula, melting point and elementary analysis of which also are listedin Table 1.

                                      TABLE 1                                     __________________________________________________________________________     ##STR5##                                                                     Prep.                                                                         Ex-                                   m.p.(°C.) or                                                                  Elementary analysis              amp.                                                                              Structure                         refractive                                                                           Calculated                                                                             Found (%)               No. R.sub.1      R.sub.2                                                                              n Molecular formula                                                                         index  C  H  N  C  H  N                 __________________________________________________________________________    3   H                                                                                           ##STR6##                                                                            0 C.sub.56 H.sub.87 N                                                                       41-42  86.87                                                                            11.32                                                                            1.81                                                                             87.12                                                                            11.43                                                                            1.77              4   H                                                                                           ##STR7##                                                                            0 C.sub.55 H.sub.86 N.sub.2                                                                 55-56  85.21                                                                            11.18                                                                            3.61                                                                             85.03                                                                            11.37                                                                            3.51              5   H                                                                                           ##STR8##                                                                            2 C.sub.58 H.sub.91 N.HCl                                                                   103-105                                                                              83.05                                                                            11.05                                                                            1.67                                                                             83.15                                                                            11.18                                                                            1.61              6   CH.sub.3                                                                                    ##STR9##                                                                            0 C.sub.57 H.sub.89 N                                                                       37-38  86.84                                                                            11.38                                                                            1.78                                                                             86.94                                                                            11.38                                                                            1.75              7   CH.sub.3                                                                                    ##STR10##                                                                           1 C.sub.58 H.sub.91 N.HCl.H.sub.2 O                                                         87-89  81.30                                                                            11.06                                                                            1.64                                                                             81.67                                                                            10.86                                                                            1.63                   ##STR11##                                                                                  ##STR12##                                                                           0 C.sub.106 H.sub.167 N                                                                     n.sub.D.sup.25 = 1.5213                                                              87.47                                                                            11.56                                                                            0.96                                                                             87.58                                                                            11.60                                                                            0.90              9                                                                                  ##STR13##                                                                                  ##STR14##                                                                           2 C.sub.108 H.sub.171 N.HCl.2H.sub.2 O                                                      40-43  83.36                                                                            11.40                                                                            0.90                                                                             83.01                                                                            11.18                                                                            0.82              __________________________________________________________________________

PREPARATIVE EXAMPLES 10 TO 13

To a solution of 3-aminomethylpyridine (25 g.) in ethanol (100 ml) asolution of decaprenyl bromide (30 g.) in chloroform was added dropwiseat room temperature for 1 hour with stirring, which was continued forfurther 3 hours. The resulting reaction mixture after cooling was addedwith a 2 N sodium hydroxide aqueous solution (100 ml) and extracted withisopropylether. The liquid extract obtained was washed with water andsaturated saline, dried over anhydrous sodium sulfate and thenconcentrated under reduced pressure. The residue (21 g.) was purified bycolumn chromatography using silica gel (200 g.). Elution was carried outwith a mixture of 20% ethylacetate 80% hexane.3-Didecaprenylaminomethylpyridine (Ca. 1.38 g.) was obtained as an oilysubstance.

The above-said column was further eluted with a mixture of 20%ethanol/80% ethylacetate. The fraction (8.6 g.) was recrystallized fromacetone to recover crystalline 3-decaprenylaminomethylpyridine (7.8 g.).

Following the substantially same procedures as mentioned above,2-(2-decaprenylamino)-ethylpyridine,2-(2-didecaprenylamino)-ethylpyridine and3-(N-decaprenyl-N-methylamino)-methylpyridine were obtained.

The physicochemical properties of these compounds are indicated in Table2.

                                      TABLE 2                                     __________________________________________________________________________     ##STR15##                                                                    Prep.                                m.p.(°C.) or                                                                   Elementary analysis              Examp.                                                                             Structure               Molecular                                                                             refractive                                                                            Calculated                                                                             Found (%)               No.  R.sub.1        R.sub.2                                                                              n formula index   C  H  N  C  H  N                 __________________________________________________________________________    10-1                                                                                ##STR16##                                                                                    ##STR17##                                                                           1 C.sub.106 H.sub.168 N.sub.2                                                           n.sub.D.sup.25.5 = 1.5173                                                             86.58                                                                            11.51                                                                            1.91                                                                             86.46                                                                            11.60                                                                            1.86              10-2 H                                                                                             ##STR18##                                                                           1 C.sub.56 H.sub.88 N.sub.2                                                             28.1-29.2                                                                             85.21                                                                            11.24                                                                            3.55                                                                             85.04                                                                            11.34                                                                            3.33              11   H                                                                                             ##STR19##                                                                           2 C.sub.57 H.sub.90 N.sub.2.1/2H.sub.2                                                  28.2-28.6                                                                             84.28                                                                            11.29                                                                            3.45                                                                             84.52                                                                            11.33                                                                            3.29              12                                                                                  ##STR20##                                                                                    ##STR21##                                                                           2 C.sub.107 H.sub.170 N.sub.2                                                           n.sub.D.sup.25.5 = 1.5162                                                             86.57                                                                            11.54                                                                            1.89                                                                             86.52                                                                            11.66                                                                            1.83              13   CH.sub.3                                                                                      ##STR22##                                                                           1 C.sub.57 H.sub.90 N.sub.2                                                             n.sub.D.sup.25.5 = 1.5161                                                             85.21                                                                            11.29                                                                            3.49                                                                             85.11                                                                            11.37                                                                            3.30              __________________________________________________________________________

PREPARATIVE EXAMPLE 14

To a solution of N,N-dimethylbenzylamine (10 g.) in ethanol (100 ml),decaprenyl bromide (20 g.) was added dropwise at room temperature for 30minutes. Stirring is continued for 1.5 hours at room temperature. Theresulting reaction mixture is extracted with isopropylether, and theextracted phase was washed with water, dried over anhydrous sodiumsulfate and concentrated under reduced pressure. The residue (22.0 g.)obtained was purified by column chromatography on a silica gel. Then,through the column the mixtures of ethylacetate/chloroform with aconcentration gradient of 1 to 10% ethylacetate. The eluted fraction(18.6 g.) was dissolved in acetone and the resulting solution wasallowed to stand overnight in a refrigerator to recover crystallineN-benzyl-decaprenylammonium bromide (15.8 g.) m.p. 51.2°-53.1° C.

Physiological effects of the compounds of the present invention areillustrated below in detail.

(1) INTERFERON INDUCING ACTIVITY TEST

Each test compound suspended in water with a surfactant wasintraperitoneally administered to each group consisting of 5ICR femalemice weighing about 25 g. Twenty hours after administration, blood wascollected from the mice and serum was separated therefrom to obtain aserum interferon. The following steps were taken in order to determinepotency of the serum interferon thus induced. L-929 cells derived frommice and incubated previously in a monolayer was brought into contactwith the test serum solution diluted 10 times, incubated overnight at37° C. in an incubator placed in carbon dioxide atmosphere and thedilute test serum solution was removed therefrom. Thereafter, the cellswere inoculated with vesicular stomatitis virus and placed on a tissueculture medium containing 1% agar. After incubation at 37° C. for 24hours, the cells were dyed with neutral red solution diluted to anappropriate concentration to count the number of plaques formed thereonand thereby to calculate the plaque inhibition rate in each of the testgroups against a group to which no test compound had been administered.The plaque inhibition rate of each test compound is shown in Table 3.

(2) EFFECT ON MICE INFECTED WITH VACCINIA VIRUS

Groups, each consisting of 10 ICR female mice, were intravenouslyinjected vaccinia virus (DIE strain) from the vein of tail. On the 8thday after the inoculation, the number of lesions in form of small pockson the tail surface was counted after dyeing the tail with ethanolsolution containing 1% fluorescein and 0.5% methylene blue. In thistest, each test compound was administered intraperitoneally to the miceon the day just before inoculation of the virus, whereby antivirusactivity of the test compound was evaluated in terms of inhibition oftail lesions as calculated in each test group against a group to whichno test compound had been administered.

The rate of tail lesion inhibition of each test compound is shown inTable 3.

(3) EFFECT ON MICE INFECTED WITH INFLUENZA VIRUS

Groups, each consisting of 10 ICR female mice weighing about 25 g. werechallenged by inhalation of neblyzed influenza virus A/PR-8. A solutionof each test compound in an aqueous solution containing a surfactant wasintraperitoneally administered to the mice 24 hours and 3 hours beforethe virus infection, and 5 times every other day from the second dayafter the infection. The mice that survived 21 days after the challengewere regarded as survivors, and survival rate was obtained according tothe following equation. ##EQU1##

                                      TABLE 3                                     __________________________________________________________________________                      Inhibition of tail                                                                     Survival rate                                                                          Plaque                                                      lesion   (Prevention from                                                                       inhibition                                              Dose                                                                              (Prevention from                                                                       influenza                                                                              (Serum                                                  (i.p.)                                                                            vaccinia infection)                                                                    injection)                                                                             interferon)                               Test compound mg/kg                                                                             %        %        %                                         __________________________________________________________________________    N--methyl-N--phenyl-                                                                        50  29.3     50        3.8                                      decaprenylamine                                                               N--benzyl-decaprenylamine                                                                   50  73.2     50       98.6                                      hydrochloride                                                                 N--methyl-N--benzyl-                                                                        50  45.3     70       24.2                                      decaprenylamine                                                               hydrochloride                                                                 N--phenethyl- 50  51.6     80       20.4                                      decaprenylamine                                                               hydrochloride                                                                 3-didecaprenyl-                                                                             50   6.4     --       --                                        aminomethylpyridine                                                           3-decaprenylamino-                                                                          50  47.4     70       --                                        methylpyridine                                                                2-(2-decaprenylamino)                                                                       50  81.1     90       --                                        ethylpyridine                                                                 2-(2-didecaprenylamino)-                                                                    50  34.5     --       --                                        ethylpyridine                                                                 3-(N--decaprenyl-N--                                                                        50  47.9     10       --                                        methylamino)methylpyridine                                                    Benzyl-decaprenyl-                                                                          50  22.8     --       --                                        dimethylammonium bromide                                                      Amantadine hydrochloride                                                                    50  --       40       --                                        (Control)                                                                     __________________________________________________________________________

(4) TOXICITY

In order to investigate acute toxicity of the compounds of the presentinvention, 50% lethal dose of each compound was obtained by using ddYmale mice weighing 20-25 g. From the results shown in Table 4, it isunderstood that the compounds had high safety margin by intraperitonealadministration.

                  TABLE 4                                                         ______________________________________                                                     50% Lethal dose (mg/kg)                                                         Intravenously                                                                            Intraperitoneally                                   Test compound  administered                                                                             administered                                        ______________________________________                                        N--methyl-N--phenyl-                                                                         180        >500                                                decaprenylamine                                                               N--benzyl-     220         315                                                decaprenylamine                                                               hydrochloride                                                                 N--methyl-N--benzyl-                                                                         377        >500                                                decaprenylamine                                                               hydrochloride                                                                 N--phenethyl-  110        >500                                                decaprenylamine                                                               hydrochloride                                                                 3-decaprenylamino-                                                                           --         >500                                                methylpyridine                                                                2-(2-decaprenylamino)-                                                                       --         >500                                                ethylpyridine                                                                 ______________________________________                                    

As is clear from the foregoing test results, the active ingredients ofthe present invention have interferon inducing activity in vivo and arelow in toxicity with showing excellent antiviral activity. In the lightof the fact that the strict correlation of interferon activity with theindividual antivirus activities is not always observed for the presentingredients, there is considered also a possibility that the antivirusactivities of said ingredients at biological level are concerned notonly in interferon but also in other defensive mechanism of host.Accordingly, when the active ingredients of the present invention areused for treatment of virus-infected diseases, they are administered topatients by such techniques involving oral, inhalant, or the likeadministration as well as subcutaneous, intramascular and intravenousinjection. According to the condition of patient such as age, symptomand route by which the ingredient is administered, the active ingredientof the present invention is used in a dose of 0.5-20 mg/kg, preferably3-5 mg/kg several times (2-4 times) per day.

The active ingredients of the present invention can be formulated intocompositions for medication, for example, tablets, capsules, granules,powder, liquid preparation for oral use, eye lotions, suppositories,ointments, injections and the like.

When the present active ingredients are orally administered, they may beformulated into tablets, capsules, granules or powder. These solidpreparations for oral use may contain commonly used excipients, forexample, silicic anhydride, metasilicic acid, magnesium alginate,synthetic aluminum silicate, lactose, cane sugar, corn starch,microcrystalline cellulose, hydroxypropylated starch or glycine, and thelike; binders, for example, gum arabic, gelatin, tragacanth,hydroxypropyl cellulose or polyvinylpyrrolidone; lubricants, forexample, magnesium stearate, talc or silica; disintegrating agents, forexample, potato starch and carboxymethyl cellulose; or wetting agents,for example, polyethylene glycol, sorbitan monooleate, hydrogenatedcastor oil, sodium laurylsulfate. In preparing soft capsules, inparticular, the present active ingredients may be formulated bydissolving or suspending them in commonly used oily substrates such assesame oil, peanut oil, germ oil, fractionated coconut oil such asMiglyol®, or the like. Tablet or granule preparations may be coatedaccording to the usual method.

Liquid preparation for oral use may be in the form of aqueous or oilyemulsion or syrup, or alternatively in the form of dry product which canbe re-dissolved before use by means of a suitable vehicle. To theseliquid preparations, there may be added commonly used additives, forexample, emulsifying aids such as sorbitol syrup, methyl cellulose,gelatin, hydroxyethyl cellulose and the like; or emulsifiers, forexample, lecithin, sorbitan monooleate, hydrogenated castor oil,non-aqueous vehicles, for example, fractionated coconut oil, almond oil,peanut oil and the like; or antiseptics, for example, methylp-hydroxybenzoate, propyl p-hydroxybenzoate or sorbic acid. Further,these preparations for oral use may contain, if necessary,preservatives, stabilizers and the like additives.

In case where the present active ingredients are administered in theform of non-oral suppository, they may be formulated according to theordinary method using oleophilic substrates such as cacao oil orWitepsol®, or may be used in the form of rectum capsule obtained bywrapping a mixture of polyethylene glycol, seame oil, germ oil,fractionated coconut oil and the like in a gelatin sheet. The rectumcapsule may be coated, if necessary, with waxy materials.

When the present active ingredients are used in the form of injection,they may be formulated into preparations of oil solution, emulsifiedsolution or aqueous solution, and these solutions may contain commonlyused emulsifiers, stabilizers or the like additives.

According to the method of administration, the above-mentionedcompositions can contain the present active ingredients in an amount ofat least 1%, preferably 5 to 50%.

The procedure of formulating the present active ingredients into variouspreparations is illustrated below with reference to PharmaceuticalExamples.

PHARMACEUTICAL EXAMPLE 1 Hard capsule preparations for oral use

A mixture of 25 g. of N-benzyl-decaprenylamine hydrochloride and 7.5 g.of polyoxyethylene castor oil in acetone was mixed with 25 g. of silicicanhydride. After evaporation of the acetone, the mixture was mixedfurther with 5 g. of calcium carboxymethylcellulose, 5 g. of cornstarch, 7.5 g of hydroxypropylcellulose and 20 g. of microcrystallinecellulose, and 30 ml of water was added thereto and kneaded to give agranular mass. The mass was pelletized by means of a pelletizer (ECKpelletter of Fuji Paudal Co., Japan) equipped with No. 24 mesh (B.S.)screen to obtain granules. The granules were dried to less than 5%moisture content and screened with No. 16 mesh (B.S.) screen. Thescreened granules were capsuled by means of a capsule filling machine soas to be contained in an amount of 190 mg per capsule.

PHARMACEUTICAL EXAMPLE 2 Soft capsule preparations for oral use

A homogeneous solution was prepared by mixing 50 g. ofN-methyl-N-benzyl-decaprenylamine hydrochloride with 130 g. ofpolyethylene glycol (Macrogol 400). Separately, a gelatin solution wasprepared which contained 93 g. of gelatin, 19 g. of glycerine, 10 g. ofD-sorbitol, 0.4 g of ethyl p-hydroxybenzoate, 0.2 g. of propylp-hydroxybenzoate and 0.4 g. of titanium oxide and which was used as acapsule film forming agent. The previously obtained solution, togetherwith the capsule film forming agent, was treated with a manual type flatpunching machine to obtaine capsules each having the contents of 180 mg.

PHARMACEUTICAL EXAMPLE 3 Injections

A mixture of 5 g. of N-methyl-N-phenyl-decaprenylamine hydrochloride, anappropriate amount of peanut oil and 1 g. of benzyl alcohol was made atotal volume of 100 cc by addition of peanut oil. The solution wasportionwise poured in an amount of 1 cc under asepsis operation into anampule which was then sealed.

PHARMACEUTICAL EXAMPLE 4 Injections

A mixture of 1.0 g of N-phenethyl-decaprenylamine hydrochloride, 5.0 g.of Nikkol HCO 60 (a tradename) (hydrogenated castor oilpolyoxyethylene-60 mols-ether), 20 g. of propylene glycol, 10 g. ofglycerol and 5.0 g. of ethyl alcohol was mixed with 100 ml of distilledwater and stirred. Under asepsis operation, the solution was portionwisepoured in an amount of 1.14 ml into an ampule which was then sealed.

What we claim is:
 1. ##STR23## wherein n represents an integer of 0-2,R₁ represents hydrogen atom, a lower alkyl group or decaprenyl group,and R₂ represents the phenyl group, and pharmaceutically acceptable acidaddition salts thereof.
 2. N-benzyl-decaprenylamine hydrochloride. 3.N-benzyl-didecaprenylamine hydrochloride.